ABCB5-positive stem cells for LSCD therapy

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ABSTRACT- limited to 30 lines The corneal epithelium is a rapidly self-renewing epithelial surface essential for maintaining a clear cornea and normal vision. The limbus contains a small subpopulation of limbal stem cells (LSCs) that continually repopulate the corneal epithelium and patients with limbal stem cell deficiency (LSCD) are unable to regenerate the corneal epithelium, resulting in "conjunctivalization" that ultimately leads to blindness. We previously identified that the ABCB5 gene is expressed by LSCs in both mouse and human tissues and that normal function of ABCB5+ LSCs is required for corneal development, homeostasis, and repair. Moreover, ABCB5 is a cell surface protein and specific monoclonal antibodies are capable of isolating pure ABCB5+ LSCs for study and transplantation. Research from our prior grants supported the first clinical trials using purified in vitro-expanded human allogeneic ABCB5+ LSCs for patients with bilateral LSCD (ClinicalTrials.gov: Identifier NCT03549299). Despite our success, the use of donor allogeneic ABCB5+ LSC transplants is suboptimal because it requires immunosuppressive treatment to prevent immune rejection of donor cells. Allogeneic donor ABCB5+ LSCs were used because LSCD patients lack a source of healthy autologous ABCB5+ LSCs since both eyes are involved. To solve this problem and to treat bilateral LSCD patients with autologous ABCB5+ LSCs, this grant renewal will study three novel approaches. The first approach uses transplantation of ABCB5+ iLSCs (induced LSCs derived from iPSCs generated from skin) using full reprogramming of autologous skin cells, using the Yamanaka genes OSKM (Oct4, Sox2, Klf4, c-Myc) to produce human induced pluripotent stem cells (iPSCs) that are then differentiated in vitro into 2D eye-like organoids that remarkably contain ABCB5+ LSCs (Watanabe et al., iScience, 2021). The second approach uses transplantation-independent restoration and expansion of residual dysfunctional or quantity-deficient LSCs, using in vivo partial epigenetic reprogramming, a new form of reprogramming discovered by Co-PI Dr. Bruce Ksander and collaborators (Lu et al., Nature, 2020), which uses transient expression of three Yamanaka genes, OSK, to restore and rescue injured dysfunctional cells in vivo. The third approach uses niche-produced LAMA5 ligand-based signaling activation of the BCAM molecular pathway that we just discovered is critical to ABCB5+ LSC and Transient Amplifying Cell (TAC) expansion (Sasamoto et al. Cell Reports, 2022, in press), with resultant stem cell proliferation programs that enhance corneal regeneration. Thus, our hypothesis is that it is possible to manufacture and expand autologous ABCB5+ LSCs from patients with bilateral LSCD using (i) full reprogramming in vitro, (ii) partial epigenetic reprogramming in vivo, and (iii) niche-produced LAMA5 ligand-based activation of LSCs through specific BCAM/ABCB5 signal transduction pathways. These autologous ABCB5+ LSC will fully regenerate and maintain a normal limbus and corneal epithelium. This hypothesis will be tested in three independent specific aims that are thematically related.